Phthalide compounds and their production process

ABSTRACT

This invention provides processes for producing phthalide compounds which comprise cultivating Phanerochaete avelutina FERM BP-4787 and then isolating phthalide compounds from the fermentation broth.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of pending International PatentApplication No. PCT/IB96/00820, filed on Oct. 02, 1995, entitled"Phthalide Compounds and their Production Process", which is acontinuation in-part of International Patent Application No.PCT/JP94/01962, filed on Nov. 21, 1994, entitled "Phthalide Compoundsand their Production Process", now abandoned.

1. Technical Field

This invention relates to novel phthalide compounds, and particularly tonovel phthalide compounds produced by fermentation of a microorganismwhich has been deposited as FERM BP-4787. This invention also relates toprocesses for producing the phthalide compounds, and a pharmaceuticalcomposition comprising the same, which is useful in the treatment ofdiseases, disorders and adverse conditions caused by Helicobacter pyloriand is particularly useful in the treatment of gastroduodenal disorders,diseases and adverse conditions caused thereby.

2. Background Art

Gastric and duodenal ulcers affect a significant portion of the humanpopulation worldwide. Currently, the usual treatment for both gastricand duodenal ulcers involves treatment of the patient with histamine H₂receptor antagonists (H₂ blockers). While generally effective in healingulcers, ulcer relapse occures in up to 90% of patients within a year ofdiscontinuing the H₂ blocker therapy. Thus, patients must continue thetreatment for many years or risk a recurrence of the ulcer. It is nowknown that ulcer healing drugs such as colloidal bismuth subcitrate(CBS) are helicobactericidal and that such CBS is used in combinationwith H₂ blockers to treat ulcers. Additionally, CBS, an H₂ blocker andamoxicillin have been used in combination to treat ulcer patients.

Helicobacter pylori has been recently demonstrated to be a majorcausative agent in gastric and duodenal ulcers and other gastroduodenaldisorders, diseases and adverse conditions. Thus, antibiotic therapy toeliminate Helicobacter pylori from the gastroduodenal tract would removethe root cause of said gastroduodenal disorders, diseases and adverseconditions and eliminate the need for an ulcer patient to continue longand costly treatment with H₂ blockers and the like. None of theforegoing treatments are capable of 100% eradication of Helicobacterpylori

Therefore, it would be desired to provide a compound having an excellenthelicobactericidal activity.

The object of the present invention is to provide novel phthalidecompounds having a excellent helicobactericidal activity and apharmaceutical composition comprising the same. Another object of thepresent invention is to provide processes for producing the novelphthalide compounds.

BRIEF DISCLOSURE OF THE INVENTION

Accordingly, the present invention provides phthalide compounds of theformula: ##STR1## wherein R is a group of the formula: ##STR2## --(CH₂)₃--C(═O)--(CH₂)₂ --C(═O)--CH₃, --(CH₂)₅ --C(═O)--(CH₂)₂ --C(═O)--CH₃,--(CH₂)₆ --CH(OH)--CH₃ or --(CH₂)₆ --C(═O)--CH₃.

Among the compounds of formula (I) of the present invention, prefercompound of formula (I) wherein R is: ##STR3##

Further, the present invention provides processes for producing thephthalide compounds of formula (I), which comprise cultivating amicroorganism having identifying characteristics of FERM BP-4787, or amutant or recombinant form thereof.

Also, the present invention provides a pharmaceutical composition foruse in the treatment of Helicobacter pylori-induced disorders, diseasesor adverse conditions caused thereby, such as gastroduodenal disordersincluding gastric ulcer, duodenal ulcer and gastric cancer, whichcomprises compounds of formula (I) and a pharmaceutically-acceptablecarrier.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism used in this invention is a strain of Phanerochaetevelutina FPL 5362 which was obtained from the Forest Products Laboratoryof the United States Department of Agriculture (Madison, Wis. It wasdeposited under the accession number FERM BP-4787 to National Instituteof Bioscience and Human-Technology, Agency of Industrial Science andTechnology (located at 1-3, Higashi 1-chome, Tsukuba, Ibaraki 305,Japan) under the Budapest Treaty on Aug. 25, 1994. The taxonomicalproperties of this strain have been reported by Burdsall, H. H., Jr.(Mycologia Memoir, 10, 133-139, 1985), describing that the strain is thebasidiomycete Panerochaete velutina.

In this invention, a mutant or recombinant form of FERM BP-4787 havingthe ability to produce the phthalide compounds of formula (I) can bealso used. The mutant or recombinant form may be obtained by spontaneousmutation, artificial mutation with ultraviolet radiation, or treatmentwith mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine or ethylmethanesulfonate, or a cell technology method such as cell fusion, genemanipulation or the like, according to well-known methods.

According to the present invention, the phthalide compounds of formula(I) may be produced by aerobic fermentation of FERM BP-4787, or a mutantor recombinant form thereof, under conditions similar to those generallyemployed to produce bioactive compounds by fermentation.

FERM BP-4787, or a mutant or recombinant form thereof, is usuallyfermented on solid medium with an insoluble material and aqueousnutrient media. The amount of the insoluble material may range 10 to 50%(w/v). Suitable insoluble materials useful for fermentation includesand, cracked stone, wood chip and whole and broken grains, such aswheat bran, oatmeal, cracked corn, millet, etc. In this invention,cultivation of FERM BP-4787 to produce the novel phthalide compounds waspreferably carried out using such insoluble materials and aqueousnutrient media at a temperature of 20°to 35° C. for 3 to 30 days. The pHof the medium may be adjusted in the range from 4.0 to 9.0, preferablyfrom 5.0 to 7.5.

Nutrient media useful for fermentation include a source of assimilablecarbon such as sugars, starches and glycerol; and a source of organicnitrogen such as casein, enzymatic digest of casein, soybean meal,cotton seed meal, peanut meal, wheat gluten, soy flour, meat extract andfish meal. A source of growth substances such as mineral salts, sodiumchloride and calcium carbonate; and trace elements such as iron,magnesium, copper, zinc, cobalt and manganese may also be utilized withadvantageous results.

FERM BP-4787, or a mutant or recombinant form thereof, can also befermented under submerged aerobic conditions with agitation at atemperature of 15°to 40° C. for 3 to 25 days, which may be variedaccording to scale and fermentation conditions such as medium andtemperature. FERM BP-4787 is preferably fermented to produce saidphthalide compounds in aqueous nutrient media at a temperature of 20°to35° C. for 3 to 25 days. The pH of the medium may be adjusted in therange from 4.0 to 9.0, preferably from 5.0 to 7.5. Nutrient media usedfor submerged aerobic conditions include those described above. Ifexcessive foaming is encountered during fermentation, antifoam agentsuch as polypropylene glycols or silicones may be added to thefermentation medium.

The phthalide compounds thus produced may be isolated by standardtechniques such as extraction and various chromatographic techniques.The phthalide compounds, CJ-12,954, CJ-13,014, CJ-13,015, CJ-13,102,CJ-13,103, CJ-13,104 and CJ-13,108 were isolated in a substantially pureform from the fermentation mixture, and identified by variousspectroscopic techniques such as UV spectrophotometry, NMR and massspectrometries. According to the analyses, these compounds are believedto have the following chemical formulas. ##STR4##

The helicobactericidal activity of the phthalide compounds of thisinvention was determined by an agar plate dilution method using thepaper disk (8 mm, ADVANTEC) and Brucella agar medium (BBL-BectonDickinson Microbiology Systems). The phthalide compounds, CJ-12,954,CJ-13,014, CJ-13,015, CJ-13,102, CJ-13,103, CJ-13,104 and CJ-13,108showed a helicobactericidal activity. Among these compounds, CJ-12,954and CJ-13,014 showed especially high activity. When tested against othermicroorganisms such as Bacillus stearothermophilus, Micrococcus luteus,Staphylococcus aureus and Pasteurella haemolytica at the concentrationsequivalent to those used in the above test, none of the phthalidecompounds showed bactericidal activity against these microorganisms.

Accordingly, the phthalides compounds of formula (I) of the presentinvention are useful in the treatment of Helicobacter pylori-induceddisorders, diseases or adverse conditions caused thereby, such asgastroduodenal disorders including gastric ulcer, duodenal ulcer andgastric cancer. For use as a helicobactericidal agent in a mammaliansubject, especially a human subject, the phthalide compounds of formula(I) of the present invention can be administered either alone, or withan inert carrier in a pharmaceutical composition, according to standardpharmaceutical practice. The phthalide compounds of formula (I) of thisinvention may also be administered in combination with a suitable H₂blocker such as ranitidine, cimetidine, famotidine or nizatinine, aproton pump inhibitor such as omeprazole, other antibiotic such asamoxicillin, or any combination thereof. If desired, CBS may be alsoadded to the pharmaceutical composition. The compounds can be applied byparenteral or oral administration. The active ingredient may becompounded, for example, with the usual non-toxic,pharmaceutically-acceptable carriers for tablets, pellets, capsules,suppositories, solutions, emulsions, suspensions and other formssuitable for use. The carriers which can be used are water, glucose,lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea and other carriers suitable for use in manufacturingpreparations. In addition, if needed, auxiliary, stabilizing andcoloring agents and perfumes may be used. In general, the phthalidecompounds of this invention are present in such dosage forms atconcentration levels ranging 5 to 70% by weight, preferably 10 to 50% byweight.

The phthalide compounds of formula (I) of this invention can be used inmammalian subjects as helicobactericidal agents in dosages ranging from0.01 to 20 mg/kg. The dosage to be used in a particular case will varyaccording to a number of factors such as the disease state or conditionbeing treated, the potency of the individual compound beingadministered, the response of the particular subject and the route ofadministration. However, when a phthalide compound of formula (I) isused in a human patient to treat gastroduodenal disorders, the usualoral or parenteral dosage will be in the range from 0.5 to 250 mg andpreferably 5 to 250 mg, one to four times per day.

EXAMPLES

The present invention is illustrated by the following examples. However,it should be understood that the invention is not limited to thespecific details of these examples. Spectral and physico-chemical datawere obtained by the following instruments: UV, JASCO Ubest-30; IR,Shimadzu IR-470; NMR, JEOL JNM-GX270 updated with a LSI-11/73 hostcomputer, TH-5 tunable probe and version 1.6 software; and LREI- andHREI-MS, Hitachi M-80 with an M-003 data processing system. All NMRspectra were measured in CDCl₃ unless otherwise indicated and peakpositions are expressed in parts per million (ppm) based on the internalstandard of the CHCl₃ peak at 7.24 ppm. The peak shapes are denoted asfollows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet),q (quartet); m (multiplet), br (broad), brs (broad singlet) and sh(shoulder).

Example One

Cells from a 10- to 21-day-old petri dish of Panerochaete velutina (FERMBP-4787) grown on malt agar medium (malt extract 2.5% and agar 1.5%)were harvested and suspended in 2 ml sterile water. This suspension wasused to inoculate 50-ml tubes containing 10 ml of Medium-1 (glucose 2%,malt extract 2%, yeast extract 1.8%, maltose 2.4% and agar 0.1%, pH5.4˜5.6). The tubes were incubated at 28° C. on a shaker at 250 rpm for7 to 21 days.

Example Two

The cell suspension (2 ml) obtained by the method of Example One wasused to inoculate two 500-ml flasks containing 100 ml of Medium-1. Theflasks were incubated at 28° C. for 7 days. These flasks were used toinoculate 7.5 ml into fifteen 500-ml flasks containing 150 ml ofMedium-1. These seed cultures in the 15 flasks were used to inoculate100 ml into fifteen 6-1 fermentation vessels containing 0.5 l ofMedium-2 (glucose 1%, glycerol 3%, peptone 0.5%, NaCl 0.2%, agar 0.1%and pH 6.9˜7.1) and 200 g oatmeal. Incubation was carried out at 28° C.for 14 to 28 days.

Example Three

According to the method of Example Two, fermentation was carried out,except that the seed cultures in 12 shake flasks were used to inoculate100 ml into twelve 6l fermentation vessels containing 0.5 or 0.75l ofMedium-1 and 300 g wheat bran. Incubation was carried out at 28° C. for14 to 28 days.

Example Four

Isolation of Phthalide Compounds

To each of the twelve 6 l fermentation vessels of Example Three, 2 l ofethanol was added. The combined broth was filtered, the filtrate wasconcentrated to aqueous solution (6 l), and extracted three times with 5l of ethyl acetate. The extract was dried over anhydrous Na₂ SO₄ andevaporated to afford an oily residue (84.4 g). The oily residue wasloaded on a silica gel column and compounds were eluted with ethylacetate-n-hexane (1:3) (3 ), then ethyl acetate-n-hexane (1:1) (3).Fractions showing activity were applied separately to a Sephadex LH-20(Pharmacia trademark) column and eluted with methanol. Fractionscontaining phthalide compounds were further applied to a Chemcosorb5ODS-UH (Chemco trademark) column (20×250 mm) and eluted withacetonitrile-water (13:7) for 120 min at a flow rate of 10 ml/min. Thedetection was made by UV absorbance at 230 nm.

The eluted peaks showing activity were collected to yield the compoundsCJ-12,954 (15.2 mg), CJ-13,014 (14.3 mg), CJ-13,015 (7.8 mg), CJ-13,102(21.1 mg), CJ-13,103 (4 mg), (CJ-13,104 (14.1 mg) and CJ-13,108 (54 mg).The seven compounds were separated by HPLC using a YMC-pack ODS AM-302(Yamamura trademark) (150×4.6 mm column) and eluted with methanol-water(3:1) for 30 min at a flow rate of 1 ml/min at 40° C. The retentiontimes (min) of the separated compounds were 10.7 (CJ-12,954), 11.4(CJ-13,014), 22.1 (CJ-13,015), 11.0 (CJ-13,102), 11.8 (CJ-13,103), 24.4(CJ-13,104) and 20.9 (CJ-13,108).

Characterization

The physicochemical properties of CJ-12,954, CJ-13,014, CJ-13,015,CJ-13,102, CJ-13,103, CJ-13,104 and CJ-13,108 are as follows.

CJ-12,954

Colorless glass; α!_(D) ²⁴ +6.0° (c 0.07, CHCl₃); HREI-MS (m/z) obs.418.2355 (calcd. for C₂₄ H₃₄ O₆, 418.2354); UV λ_(max) (MeOH, nm) 217.4(ε30,000), 257.6 (ε14,000) and 290.6 (ε5,000); IR (KBr, cm⁻¹) 2930,1754, 1606, 1489, 1457, 1433, 1333, 1214, 1155, 1105, 1051, 1027, 985,856, 836 and 688; LREI-MS (m/z) 418 (2.7%, rel. int.), 403 (1.0), 400(1.2), 385 (2.4), 374 (4.9), 363 (4.8), 361 (16.1), 320 (33.1), 318(7.8), 307 (6.5), 303 (3.5), 279 (5.3), 278 (8.8), 207 (22.8), 194(29.6), 193 (80.6), 177 (6.1), 165 (12.2), 141 (100), 112 (21.3), 85(68.1), 55 (29.1) and 41 (24.5); ¹ H NMR δ6.39 (1H, brs), 6.38 (1H,brs), 5.27 (1H, dd, J=8.1, 3.7 Hz), 4.08 (1H, m), 3.93 (3H, s), 3.90(1H, m), 3.87 (3H, s), 2.05˜1.80 (7H, m) 1.80˜1.55 (4H, m), 1.55˜1.20(9H, m) and 1.27 (3H, d, J=6.2 Hz); ¹³ C NMR δ168.5 (s), 166.6 (s),159.6 (s), 155.2 (s), 114.2 (s), 106.9 (s), 98.6 (d), 97.3 (d), 79.9(d×2), 75.8 (d), 56.0 (q), 55.9 (q), 37.3 (t), 36.5 (t), 36.1 (t), 34.8(t), 32.6 (t), 30.7 (t), 29.4 (t), 29.3 (t), 25.9 (t), 24.6 (t) and 23.0(q).

CJ-13,014

Colorless glass; α!_(D) ²⁴ +71.2° (c 0.11, CHCl₃); HREI-MS (m/z) obs.418.2355 (calcd. for C₂₄ H₃₄ O₆, 418.2354); UV λ_(max) (MeOH, nm) 217.0(ε34,000), 257.8 (ε16,000) and 290.4 (ε5,6000); IR (KBr, cm⁻¹) 2930,1753, 1607, 1489, 1457, 1432 1333, 1214, 1156, 1100, 1050, 1027, 916,894, 865, 835 and 690; LREI-MS (m/z) 418 (3.7%, rel. int.), 403 (1.2),400 (1.7), 385 (2.4), 374 (5.1), 363 (6.2) 361 (35.2), 360 (5.7), 345(1.1), 320 (19.7), 318 (8.6), 307 (8.6), 303 (4.2), 279 (4.4), 278(7.9), 207 (24.0), 194 (28.0), 193 (79.9), 191 (7.1), 177 (5.9), 165(11.4), 141 (100), 135 (7.5), 123 (4.9), 122 (6.4), 112 (24.4), 85(54.4), 67 (7.8), 57 (10.6), 56 (15.8) and 55 (26.0); ¹ H NMR δ6.39 (1H,brs), 6.38 (1H, brs), 5.27 (1H, dd, J=8.1, 3.7 Hz), 4.16 (1H, m), 4.01(1H, m), 3.93 (3H, s), 3.87 (3H, s), 2.15˜1.90 (7H, m), 1.65 (1H, m),1.55˜1.35 (6H, m), 1.35˜1.25 (6H, m) and 1.19 (3H, d, J=6.2 Hz); ¹³ CNMR δ168.5 (s), 166.6 (s), 159.6 (s), 155.2 (s), 114.7 (s), 106.9 (s),98.6 (d), 97.3 (d), 79.9 (d), 78.1 (d), 74.0 (d), 56.0 (q), 55.9 (q),35.7 (t), 35.6 (t×2), 34.8 (t), 32.2 (t), 30.2 (t), 29.4 (t), 29.3 (t),25.7 (t), 24.6 (t) and 21.1 (q).

CJ-13,015

Colorless glass; α!_(D) ²⁵ +25.6° (c 0.22, CHCl₃); HREI-MS (m/z) obs.418.2358 (calcd. for C₂₄ H₃₄ O₆, 418.2354); UV λ_(max) (MeOH, nm) 217.4(ε32,000), 257.6 (ε15,000) and 290.4 (ε5,800); IR (KBr, cm⁻¹) 2920,2850, 1757, 1697, 1612, 1599, 1493, 1465, 1426, 1412, 1350 (sh), 1333,1220, 1197, 1160, 1096, 1050, 1023, 835 and 689; LREI-MS (m/z) 418(10.3%, rel. int.), 400 (11.8), 375 (3.8), 347 (18.2), 320 (11.4), 305(70.0), 207 (36.8), 194 (28.7), 193 (100), 165 (13.7), 99 (19.3) and 43(32.3); ¹ H NMR δ66.39 (1H, brs), 6.38 (1H, brs), 5.27 (1H, dd, J=8.1,3.7 Hz), 3.93 (3H, s), 3.87 (3H, s), 2.67 (4H, m), 2.42 (2H, t, J=7.5Hz), 2.17 (3H, s), 1.95 (1H, m), 1.66 (1H, m), 1.54 (2H, m), 1.42 (2H,m) and 1.24 (10H, brs); ¹³ C NMR δ209.7 (s), 207.4 (s), 168.6 (s), 166.6(s), 159.5 (s), 155.2 (s), 106.7 (s), 98.6 (d), 97.3 (d), 80.0 (d), 55.9(q×2), 42.7 (t), 36.8 (t), 36.0 (t), 34.8 (t), 29.9 (q), 29.2 (t×4),29.1 (t), 24.6 (t) and 23.7 (t).

CJ-13,102

Colorless glass; α!_(D) ²⁵ +26.8° (c 0.81, CHCl₃); HREI-MS (m/z) obs.462.2620 (calcd. for C₂₆ H₃₈ O₇, 462.2616); UV λ_(max) (MeOH, nm) 217.2(ε24,000), 257.4 (ε11,000) and 290.4 (ε4,000); IR (KBr, cm⁻¹) 2925,2850, 1754, 1732, 1606, 1489, 1460, 1430, 1356, 1334, 1239, 1217, 1157,1102, 1052, 1027 and 835; LREI-MS (m/z) 462 (5.5%, rel. int.), 402(91.5), 363 (47.9), 345 (43.2), 207 (39.0), 194 (29.7), 193 (100), 165(13.3) and 43 (66.0); ¹ H NMR δ6.38 (2H, brs), 5.26 (1H, dd, J=7.7, 3.7Hz), 4.81 (1H, m), 3.91 (3H, s), 3.86 (3H, s), 2.42 (2H, t, J=7.5 Hz),2.11 (3H, s), 2.00 (3H, s), 2.00˜1.55 (4H, m), 1.55˜1.30 (4H, m) and1.21 (12H, brs); ¹³ C NMR δ208.0 (s), 170.9 (s), 168.5 (s), 166.6 (s),159.6 (s), 155.2 (s), 106.9 (s), 98.6 (d), 97.4 (d), 79.9 (d), 73.5 (d),55.9 (q×2), 39.5 (t), 34.8 (t), 34.2 (t), 29.9 (q), 29.3 (t×4), 29.2(t), 27.9 (t), 25.2 (t), 24.6 (t) and 21.1 (q).

CJ-13,103

Colorless glass; α!_(D) ²⁵ +26.0° (c 0.10, CHCl₃); HREI-MS (m/z) obs.446.2665 (calcd. for C₂₆ H₃₈ O₆, 446.2666); UV λ_(max) (MeOH, nm) 216.8(ε26,000), 257.4 (ε12,000) and 290.0 (ε4,600); IR (KBr, cm⁻¹) 2920,2845, 1759, 1699, 1612, 1599, 1492, 1463, 1426, 1412, 1356, 1332, 1220,1195, 1160, 1095, 1047, 1024, 834 and 690; LREI-MS (m/z) 446 (18.5%,rel. int.), 428 (19.8), 403 (6.2), 375 (21.2), 348 (13.4), 333 (100),207 (32.0), 194 (25.1), 193 (69.6), 165 (8.8), 99 (13.7), 71 (9.7), 55(109) and 43 (23.4); ¹ H NMR δ6.39 (1H, brs), 6.38 (1H, brs), 5.27 (1H,dd, J=7.7, 3.7 Hz), 3.93 (3H, s), 3.87 (3H, s), 2.66 (4H, m), 2.42 (2H,t, J=7.5 Hz), 2.16 (3H, s), 1.94 (1H, m), 1.64 (1H, m), 1.52 (2H, m),1.41 (2H, m) and 1.22 (14H, brs).

CJ-13,104

Colorless glass; α!_(D) ²⁵ +36.1° (c 0.41, CHCl₃); HREI-MS (m/z) obs.406.2713 (calcd. for C₂₄ H₃₈ O₅, 406.2716); UV λ_(max) (MeOH, nm) 216.4(ε27,000), 257.6 (ε12,000) and 289.4 (ε4,400); IR (KBr, cm-1) 2915,2845, 1755, 1736, 1611, 1599, 1493, 1462, 1426, 1336, 1220, 1197, 1160,1097, 1052, 1026, 983, 936, 835 and 690; LREI-MS (m/z) 406 (1.2%, rel.int.), 388 (21.2), 362 (93.1), 207 (53.9), 194 (35.6), 193 (100), 165(13.5), 55 (16.5), 45 (39.6) and 41 (14.9) ¹ H NMR δ6.38 (2H, m), 5.26(1H, dd, J=7.7, 3.7 Hz), 3.92 (3H, s), 3.86 (3H, s), 3.77 (1H, m), 2.20(1H, br), 1.95 (1H, m), 1.65 (1H, m), 1.50˜1.35 (4H, m), 1.22 (18H, brs)and 1.16 (3H, d, J=6.2 Hz); ¹³ C NMR δ168.5 (s), 166.6 (s), 159.6 (s),155.2 (s), 106.9 (s), 98.6 (d), 97.4 (d), 79.9 (d), 68.2 (d), 56.0 (q),55.9 (q), 39.3 (t), 34.8 (t), 29.6 (t×5), 29.5 (t), 29.4 (t), 29.3 (t),25.7 (t), 24.6 (t) and 23.4 (q).

CJ-13,108

Colorless glass; α!_(D) ²⁵ +44.8° (c 0.50, CHCl₃); HREI-MS (m/z) obs.404.2557 (calcd. for C₂₄ H₃₆ O₅, 404.4559); UV λ_(max) (MeOH, nm) 217.4(ε30,000), 257.2 (ε14,000) and 290.2 (ε5,200); IR (KBr, cm⁻¹) 2915,2845, 1756, 1707, 1611, 1600, 1492, 1464, 1425, 1355, 1333, 1219, 1195,1159, 1099, 1050, 1023, 995, 974, 833 and 690; LREI-MS (m/z) 404 (58.7%,rel. int.), 347 (81.5), 207 (43.8), 194 (31.1), 193 (100), 165 (13.4),58 (15.5) and 43 (42.8); ¹ H NMR δ6.36 (2H, brs), 5.23 (1H, dd, J=7.7,3.7 Hz), 3.89 (3H, s), 3.84 (3H, s), 2.36 (2H, t, J=7.3 Hz), 2.07 (3H,s), 1.91 (1H, m), 1.64 (1H, m), 1.50 (2H, m), 1.38 (2H, m) and 1.18(16H, brs); ¹³ C NMR δ209.3 (s), 168.4 (s), 166.6 (s), 159.5 (s), 155.1(s), 106.8 (s), 98.5 (d), 97.4 (d), 79.9 (d), 55.9 (q), 55.8 (q), 43.7(t), 34.7 (t), 29.7 (q), 29.4 (t×3), 29.3 (t×3), 29.2 (t), 29.0 (t),24.5 (t) and 23.7 (t).

We claim:
 1. A phthalide compound of the formula: ##STR5## wherein R isa group of the formula: ##STR6## --(CH₂)₃ --C(═O)--(CH₂)₂ --C(═O)--CH₃,--(CH₂)₅ --C(═O) --(CH₂)₂ --C(═O)--CH₃, --(CH₂)₆ --CH(OH)--CH₃ or--(CH₂)₆ --C(═O)--CH₃.
 2. A phthalide compound according to claim 1,wherein R is: ##STR7##
 3. A process for producing phthalide compoundsaccording to claim 1, which comprises cultivating a microorganism havingthe identifying characteristics of Panerochaete velutina FERM BP-4787,or a mutant or recombinant form thereof.
 4. A process according to claim3, which further comprises the subsequent step of isolating saidphthalide compounds from the fermentation broth.
 5. A pharmaceuticalcomposition for use in the treatment of Helicobacter pylori induceddisorders, diseases or adverse conditions caused thereby, whichcomprises a compound according to claim 1 and apharmaceutically-acceptable carrier.